Ischemic vascular accident revealing hypoplasia of the internal carotid artery in a narrowed bony canal: A case report.

Hypoplasia of the internal carotid artery is a rare congenital abnormality that can present with an ischemic stroke or transient ischemic attacks. We present the case of a 17-year-old male who presented with right hemiparesis and dysarthria. The imaging revealed hypoplasia of the left internal carotid artery and narrowing of the left carotid duct. The patient was managed conservatively. This case highlights the importance of considering ICA hypoplasia as a cause of ischemic stroke in patients with a narrowed osseous canal. Early diagnosis and management can help prevent recurrent strokes.

Multiplex reverse transcription recombinase polymerase amplification combined with lateral flow biosensor for simultaneous detection of three viral pathogens in cattle.

Bovine viral diarrhea virus (BVDV), bovine epidemic fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) cause respiratory symptoms in cattle. The absence of rapid, precise, and easily accessible diagnostic methods poses difficulties for herders and veterinary epidemiologists during outbreaks of major infectious animal diseases. Considering the mixed infection of viruses, a multiple-detection method, reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), was established to simultaneously detect the three pathogens. This technique is based on the specific binding of three differently labeled RT-RPA products (DNA sequences) to antibodies on the three test lines of the LFB, achieving multiplex detection through the presence or absence of coloration on the LFB test lines. The fluorescence values of the LFB test lines are recorded by a test strip reader. The mRT-RPA-LFB assay completes detection at a constant temperature of 41 °C within 33 min. The limits of detection (LODs) for BVDV, BEFV and BRSV were 2.62 × 101, 2.42 × 101 and 2.56 × 101 copies/µL, respectively. No cross-reactivity was observed with the other six bovine viruses. The developed method showed satisfactory intra- and inter-assay precision, and the average coefficients of variation were ranged from 2.92 % to 3.99 %. The diagnostic sensitivity and specificity were 98.11 % and 100 %, respectively, which were highly consistent with the RT-qPCR assay, and the kappa value was 0.988 (95 % confidence interval, CI). In general, the mRT-RPA-LFB assay has the potential to become a powerful tool for rapid screening of cattle diseases because of its advantages such as fast detection speed, convenient operation, strong specificity, and high sensitivity.

Voltammetric and flow amperometric determination of drug guaifenesin in pharmaceutical and biological samples using screen-printed sensor with boron doped diamond electrode.

New voltammetric and flow amperometric methods for the determination of guaifenesin (GFE) using a perspective screen-printed sensor (SPE) with boron-doped diamond electrode (BDDE) were developed. The electrochemical oxidation of GFE was studied on the surface of the oxygen-terminated BDDE of the sensor. The GFE provided two irreversible anodic signals at a potential of 1.0 and 1.1 V (vs. Ag|AgCl|KCl sat.) in Britton-Robinson buffer (pH 2), which was chosen as the supporting electrolyte for all measurements. First, a voltammetric method based on differential pulse voltammetry was developed and a low detection limit (LOD = 41 nmol L-1), a wide linear dynamic range (LDR = 0.1-155 µmol L-1), and a good recovery in the analysis of model and pharmaceutical samples (RSD <3.0 %) were obtained. In addition, this sensor demonstrated excellent sensitivity and reproducibility in the analysis of biological samples (RSD <3.2 %), where the analysis took place in a drop of serum (50 µL) without pretreatment and additional electrolyte. Subsequently, SP/BDDE was incorporated into a flow-through 3D printed electrochemical cell and a flow injection analysis method with electrochemical detection (FIA-ED) was developed, resulting in excellent analytical parameters (LOD = 86 nmol L-1, LDR = 0.1-50 µmol L-1). Moreover, the mechanism of electrochemical oxidation of GFE was proposed based on calculations of HOMO spatial distribution and spectroelectrochemical measurements focused on IR identification of intermediates and products.

Development of aptamer-based lateral flow devices for rapid detection of SARS-CoV-2 S protein and uncertainty assessment.

The outbreak and spread of COVID-19 have highlighted the urgent need for early diagnosis of SARS-CoV-2. Nucleic acid testing as an authoritative tool, is cumbersome, time-consuming, and easy to cross-infect, while the available antibody self-testing kits are deficient in sensitivity and stability. In this study, we developed competitive aptamer-based lateral flow devices (Apt-LFDs) for the quantitative detection of SARS-CoV-2 spike (S) protein. Molecular docking simulation was used to analyze the active binding sites of the aptamer to S protein, guiding complementary DNA (cDNA) design. Then a highly efficient freezing strategy was applied for the conjugation of gold nanoparticles (AuNPs) and DNA probes. Under optimal conditions, the linear range of the constructed Apt-LFDs was 0.1-1 µg/mL, and the limit of detection (LOD) was 51.81 ng/mL. The cross-reactivity test and stability test of the Apt-LFDs showed good specificity and reliability. The Apt-LFDs had recoveries ranging from 89.45 % to 117.12 % in pharyngeal swabs. Notably, the uncertainty of the analytical result was evaluated using a "bottom-up" approach. At a 95 % confidence level, the uncertainty report of (453.37±54.86) ng/mL with k = 2 was yielded. Overall, this study provides an important reference for the convenient and reliable detection of virus proteins based on LFDs.

Highly sensitive detection of carbendazim in agricultural products using colorimetric and photothermal lateral flow immunoassay based on plasmonic gold nanostars.

The wide use and high toxicity of carbendazim (CBD) in agriculture pose unprecedented demands for convenient, sensitive, and cost-effective on-site monitoring. Herein, we propose a novel colorimetric and photothermal dual-mode lateral flow immunoassay (LFIA) based on plasmonic gold nanostars (AuNSs) for CBD detection in agricultural products. The AuNSs were synthesized via a rapid seed-mediated growth method (with growth time of ∼5 s). A stable immunoprobe was formed by adsorbing CBD antibodies onto AuNSs. This immunoprobe exhibited high conversion efficiency and sensitivity in photothermal detection with a low limit of detection (LOD) of 0.28 ng mL-1. The LOD of the colorimetric mode was higher (0.48 ng mL-1). The results of CBD detection in various agricultural products aligned well with ultra-performance liquid chromatography tandem mass spectrometry. Overall, our LFIA shows excellent sensitivity, specificity, reproducibility, and rapidness in CBD detection, and thus is a highly potential on-site platform in resource-limited environments.

Chronoamperometric flow-cell designed to be unaffected by air bubbles.

In this paper, we present a new design for a chronoamperometric flow cell in which air bubbles do not interfere with the control of potential between the working and reference electrodes. The flow-through dual-detection cell consists of two independent parts: an upper compartment containing a quiescent supporting electrolyte solution and a channel that operates under hydrodynamically controlled conditions. In this system, the working and counter electrodes can be placed directly in contact with both compartments, whereas the reference electrode can be assembled to be either isolated or in contact with the flowing stream channel. The design ensures that the potential applied to the working electrode (controlled in the upper compartment) is similar to the potential applied in the flowing channel. The performance of the proposed flow cell in generating accurate results, even in the presence of air bubbles, was evaluated through successive air-analyte-air injections. In both series where the analyte was introduced, suitable reproducibility was achieved. The robustness of the design was definitively proven by performing a series of measurements in analytical applications for the determination of hydrogen peroxide in antiseptic samples, yielding very satisfactory results.

γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

Flow Cytometry for Non-Hodgkin and Hodgkin Lymphomas.

Multiparametric flow cytometry is a powerful diagnostic tool that permits rapid assessment of cellular antigen expression to quickly provide immunophenotypic information suitable for disease classification. This chapter describes a general approach for the identification of abnormal lymphoid populations by flow cytometry, including B, T, NK, and Hodgkin lymphoma cells suitable for the clinical and research environment. Knowledge of the common patterns of antigen expression of normal lymphoid cells is critical to permit identification of abnormal populations at disease presentation and for minimal residual disease assessment. We highlight an overview of procedures for processing and immunophenotyping non-Hodgkin B- and T-cell lymphomas and also describe our strategy for the sensitive and specific diagnosis of classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and T-cell/histiocyte-rich large B-cell lymphoma.

Flow Cytometric MRD Detection in Selected Mature B-Cell Malignancies.

The quantification of submicroscopic minimal residual disease (MRD) after therapy proved to have independent prognostic significance in many mature B-cell malignancies. With the advent of routine benchtop cytometers capable of simultaneously analyzing ≥8 colors and with improved standardization, flow cytometry has become the method of choice for MRD assessments in some lymphoma entities. Herein we describe general aspects of flow cytometric standardization. Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are used as examples to explain the technical standardization of flow cytometry for MRD detection according to EuroFlow strategies. MRD data acquisition and detailed analysis in MM and CLL is a particular focus of this chapter.

Aqueous colloid flow batteries with nano Prussian blue.

Flow battery is a safe and scalable energy storage technology in effectively utilizing clean power and mitigating carbon emissions from fossil fuel consumption. In the present work, we demonstrate an aqueous colloid flow battery (ACFB) with well-dispersed colloids based on nano-sized Prussian blue (PB) cubes, aiming at expanding the chosen area of various nano redox materials and lowering the cost of chemicals. Taking advantage of the two redox pairs of PB, the developed all-PB cell employing a low-cost dialysis membrane with the synthesized PB on both sides displays an open-circuit voltage (OCV) of 0.74 V. Moreover, when paired with an organic tetra pyridine macrocycle the cell with PB as positive electrolyte exhibits an OCV of 1.33 V and a capacity fade rate of 0.039 %/cycle (0.8 %/day). Redox-active colloids exhibit enduring physicochemical stability, with no evident structural or morphological changes after extensive cycling, highlighting their potential for cost-effective and reliable ACFB energy storage.

Stress and flow inhomogeneity in shear-thickening suspensions.

HYPOTHESIS: The viscosity of dense suspensions surges when the applied stress surpasses a material-specific critical threshold. There is growing evidence that the thickening transition involves non-uniform flow and stress with considerable spatiotemporal complexity. Nevertheless, it is anticipated that dense suspensions of calcium carbonate particles with purely repulsive interactions may not conform to this scenario, as indicated by local pressure measurements with millimeter spatial resolution. EXPERIMENT: Here we utilize Boundary Stress Microscopy (BSM), a technique capable of resolving stresses down to the micron scale, to search for evidence of stress heterogeneity. In addition, we measure the flow field at the lower boundary of the suspension where the boundary stress is measured. FINDINGS: We find localized regions of high-stresses that are extended in the vorticity direction and propagate in the flow direction at a speed approximately half that of the rheometer's top plate. These high-stress regions proliferate with the applied stress accounting for the increased viscosity. Furthermore, the velocity of particles at the lower boundary of the suspension shows a significant and complex nonaffine flow that accompanies regions of high-stresses. Hence, our findings demonstrate that stress and flow inhomogeneity are intrinsic characteristics of shear-thickening suspensions, regardless of the nature of interparticle interactions.

Flow cytometry in acute myeloid leukemia and detection of minimal residual disease.

Acute myeloid leukemia (AML) is a common type of acute leukemia (AL), belonging to malignant tumors of the hematopoietic system with the characteristics of rapid disease development, control with extreme difficulties, easy recurrence, poor prognosis, and incidence rate increasing with age. The traditionally diagnostic standard of French American British (FAB), being based on the morphological examination with high human subjectivity, can no longer meet the demand of clinical diagnosis and treatment of AML. Requirements of objective accuracy and low-dose sample, have become the indispensable method for AML diagnosis and monitoring prognosis. Flow cytometry is a modern technology that can quickly and accurately detect the series, antigen distribution, differentiation stage of AML cells, minimal residual lesions after AML therapy, so as to provide the great significance in guiding clinical diagnosis, hierarchical treatment, and prognosis judgement. This article will systematically elaborate on the application of flow cytometry in the diagnosis and classification of AML, and the detection of minimal residual lesions, thereby providing reference significance for dynamic monitoring and prognostic observation of AML with different immune subtypes of FAB.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is linked to various signs of advanced aging, such as premature immunosenescence which occurs due to decline in regenerative ability of T cells. RA T cells develop a unique aggressive inflammatory senescent phenotype with an imbalance of Th17/T regulatory (Treg) cell homeostasis and presence of CD28- T cells. The phenotypic analysis and characterization of T cell subsets become necessary to ascertain if any functional deficiencies exist within with the help of transcription factor (TF) analysis. These subset-specific TFs dictate the functional characteristics of T-cell populations, leading to the production of distinct effector cytokines and functions. Examining the expression, activity, regulation, and genetic sequence of TFs not only aids researchers in determining their importance in disease processes but also aids in immunological monitoring of patients enrolled in clinical trials, particularly in evaluating various T-cell subsets [Th17 (CD3+CD4+IL17+RORγt+) cells and T regulatory (Treg) (CD3+CD4+CD25+CD127-FOXP3+) cells], markers of T-cell aging [aged Th17 cells (CD3+CD4+IL17+RORγt+CD28-), and aged Treg cells (CD3+CD4+CD25+CD127-FOXP3+CD28-)]. In this context, we propose and outline the protocols for assessing the expression of TFs in aged Th17 and Treg cells, highlighting the crucial aspects of this cytometric approach.

Differential Mobility Spectrometry-Based Cardiolipin Analysis.

Cardiolipins (CL) are special lipids in many respects. First of all, CL are composed of four fatty acids linked by two phosphatidic acids, which provide CL a unique molecular structure. Secondly, in eukaryotic cells they are specific to a single organelle, mitochondria, where they are also synthetized. CL are one of the most abundant lipid classes in mitochondria, mainly localized in the inner membrane. They are key determinants of mitochondrial health and homeostasis by modulating membrane integrity and fluidity, mitochondrial shapes, and metabolic pathways. Disturbances in mitochondrial CL composition can lead to tissue malfunction and diseases. It is therefore important to develop analytical tools to study the mitochondrial lipidome, and more particularly the CL. The method described here allows the quantification of cardiolipins at the sum composition level in isolated mitochondria or in liver tissue by flow injection analysis coupled to differential mobility spectrometry (FIA-DMS), also known as DMS-based shotgun lipidomics.

In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

Smartphone-based lateral flow immunoassay for sensitive determination of lactate dehydrogenase at the point of care.

Lactate dehydrogenase (LDH), a prevalent enzyme involved in anaerobic glycolysis, is released into body fluids following cell damage and has long been a general marker of tissue injury. However, due to its lack of selectivity and the advent of more accurate biomarkers, the clinical utility of LDH has been largely limited to confirming hemolysis. LDH has been recognized as a valuable prognostic biomarker for various cancers, making its monitoring crucial during cancer management. Traditional LDH methods include spectrophotometric analysis of NADH at 340 nm, native electrophoresis, or enzyme-linked immunosorbent assay. This study presents the first lateral flow immunoassay (LFIA) for the smartphone-based quantification of serum LDH levels at the point of care. Highly-affinity and specific antibodies have been produced, with 5 nM equilibrium dissociation constant and no cross-reactivity with human serum albumin and human immunoglobulin G. Utilizing carbon nanoparticles as signal transducers significantly enhanced the quantification limit 55-fold, compared to the conventional gold nanoparticles-based LFIA, achieving a quantification limit of 1.5 ng mL-1. The developed assay demonstrated a mean recovery rate of 115 ± 21 % when evaluating LDH-spiked serum samples. This method can be an interesting home-testing tool for monitoring cancer progression or therapy effectiveness.